Membrane protein function is regulated by changes in the lipid composition of the host bilayer. This regulation, though well-established, remains enigmatic as evident by the many terms that are used to describe its mechanistic basis: changes in bilayer fluidity; changes in bilayer compression or curvature frustration energies, which can be combined into the bilayer deformation energy; changes in lateral pressure profile or lipid packing stress; changes in bilayer free volume; changes in bilayer stiffness. Changes in bilayer fluidity cannot be a major mechanism, as they cannot account for changes in the equilibrium distribution among different protein conformations. The remaining descriptors represent different approaches to parameterize the interactions among bilayer-forming lipids and between the lipids and the embedded proteins. The objective of the proposed studies is to develop an energetic framework for describing the functional consequences of the hydrophobic coupling between membrane-spanning proteins and their host bilayer, and to evaluate how membrane protein-bilayer interactions can be manipulated pharmacologically. The project is based on the notion of an elastic bilayer, in which membrane proteins undergo conformational changes that perturb the adjacent bilayer. This perturbation incurs an energetic cost that contributes to the overall free energy difference of the protein conformational changes. The protein-bilayer hydrophobic coupling therefore causes protein function to vary with changes in lipid bilayer material properties (thickness, lipid intrinsic curvature and the bilayer elastic compression and bending moduli), and the lipid bilayer becomes an allosteric regulator of membrane protein function. Changes in bilayer elastic properties can be characterized in terms of a restoring force that pulls on the embedded channel to minimize the bilayer deformation energy. Changes in this force can be measured using bilayer-embedded for transducers. The experiments will address the following questions. How good is the elastic bilayer model in predicting the energetic cost of channel-bilayer bilayer interactions? Can it be applied to multi-component lipid bilayers? This will be examined using phospholipids (alone or in combination), which form bilayers that differ in thickness and lipid intrinsic curvature. How do small amphiphiles, such as free fatty acids, antimicrobial peptides and other membrane-active molecules alter lipid bilayer material properties and membrane protein function? This will be examined by probing how selected drugs alter bilayer properties, and relating this information to more complex systems. Can PI(4,5)P2 (and other phosphoinositides) alter membrane protein function through local changes in bilayer material properties? This will be examined using gramicidin analogues with appropriately designed phosphoinositide-binding domains. Can the channel-bilayer hydrophobic mismatch drive a lateral association between bilayer-spanning channels? This will be studied by examining the relative stabilization of double- barreled channels of different lengths imbedded in bilayers of different thicknesses.